Project Summary Castration resistant prostate cancer (CRPC) is a devastating disease with significant mortality. While recently approved second generation anti-androgen therapies such as enzalutamide have shown promising clinical response, most patients develop resistance. Signaling through the glucocorticoid hormone receptor (GR) is a primary mechanism of resistance to enzalutamide, indicating that GR is an attractive therapeutic target in prostate cancer. Unfortunately, existing antagonists of GR have partial agonist activity leading to unintended activation of a subset of GR target genes which limits their anti-tumor efficacy. To overcome this problem, we have utilized a recently developed technology termed proteolysis targeting chimera (PROTAC). PROTACs are an emerging class of therapeutics that consist of a small molecule ligand of a protein of interest tethered to a small molecule E3 ubiquitin ligase binder resulting in the forced approximation of the target protein to the cellular ubiquitination machinery and its degradation by the proteasome. We have synthesized a potent GR PROTAC, GR-9059, which induces robust GR degradation. Importantly, GR-9059 has a profound inhibitory activity on proliferation of enzalutamide resistant CRPC cell lines. In this proposal we aim to study the mechanisms and utility of GR-9059 as a novel therapeutic modality in CRPC. To this end, we first aim to investigate the molecular mechanism of its anti-proliferative activity on CRPC cell lines in the context of enzalutamide resistance. We will perform in depth molecular characterizations including unbiased transcriptomic and GR chromatin immunoprecipitation & sequencing (ChIP-Seq) experiments to identify direct targets of GR that are modulated by GR-9059. We also intend to experimentally develop cell lines resistant to GR PROTACs that should help in understanding potential mechanisms of drug resistance that are expected in the future if used in patients. To aid our understanding of potential side effects and tolerability of GR-9059, we aim to investigate the effect of GR-9059 on blood glucose level and immune system activation in mice, two major physiological effects of glucocorticoid hormones that are mediated by GR. Coupled to this we will determine maximum tolerated dose of GR-9059. GR-9059 is bioavailable in mice and induces robust degradation of GR in vivo. Hence, we will determine the anti-tumor efficacy of FR-9059 using established CRPC xenografts and novel PDX models. Finally, we will determine gene expression changes within tumors upon GR- 9059 treatment to elucidate the molecular alterations underlying the in vivo biological effects of GR-9059. In conclusion, these experiments are expected to elaborate the mechanism of action of GR-9059 and determine the potential utility of GR PROTACs as a new therapeutic modality in anti-androgen treatment resistant advanced prostate cancer. Finally, our experience with the design and synthesis of GR PROTACs with improved pharmacokinetic and pharmacodynamic properties will have broader ramifications in the synthesis of PROTACs against other targets in prostate and other types of cancer.